Regulation of transcription by repressors

Question 1

How do we know that CAP acts through the C-terminal domain of the alpha subunit?

Answer 1

Delete the C-terminal domain of the alpha subunit. In the absence of CAP, this mutant RNAP will transcribe as much RNA as wild type RNAP. However, in the presence of CAP, the mutant RNAP will not respond to the activator.

Question 2

Why should we care about the lac operon?

Answer 2

• It’s a classic: the lac operon was the first dissected complete gene ‘circuit’. Some of the regulation principles turned out to be universal.
• Bits and pieces of the lac operon circuit are widely used in research and industry: e.g. first human insulin was controlled by the lac = system.

Question 3

What does lac stand for in lac operon?

Answer 3

‘Lactose’

Question 4

Starvation for glucose is NOT enough to activate expression of the lac operon, why?

Answer 4

Because lac repressor is blocking the lac promoter, and RNAP cannot bind!

Question 5

The operon is ‘on’ only when what?

Answer 5

Lactose is present. That is, lactose INDUCES transcription of the operon

Question 6

What two levels of control is the lac operon under?

Answer 6

Cap activator and lac repressor

Question 7

What are the key differences between the CAP and lac repressor?

Answer 7

1. CAP binds DNA when it has its cAMP messenger in its pocket, whereas lac repressor dissociates from DNA when it has its inducer (all-lactose, IPTG) in its pocket
2. CAP has to specifically interact with RNAP to bring RNAP to promoter, whereas lac repressor does not have to interact with RNAP, it just has to be obstructive

Question 8

Can the lac repressor repress T7 RNA polymerase as well as E. coli RNA polymerase?

Answer 8

Yes

Question 9

To dissociate, and to let beta-galactosidase express, lacR has to bind allo-lactose, not lactose. Lactose itself is a poor inducer. But allo-lactose is a by-product of beta-galactosidase activity, and beta-gal has not yet been expressed. Where did this beta-galactosidase come from?

Answer 9

Any protein-DNA interaction has a lifetime (half-life). For lacR-operator interaction, the half-life is ~5 minutes. So, lacR falls off every ~5 minutes, and within 0.1 seconds binds back. As soon as lacR falls off (‘leakage’), CAP recruits RNAP, and RNAP makes 1 mRNA. 1 mRNA is enough to maintain basal levels of beta-gal (5-10 molecules per cell), which will make allo-lactose.

Question 10

Give a peculiar feature of lacR

Answer 10

The very fast rate of binding to the operator sequence

Question 11

What does 10^10 M^-1 s^-1 mean?

Answer 11

At 1M concentration, the operator will get occupied within 10^-10 seconds (0.1 billionths of a second)

Question 12

How many molecules of lacR does E. coli normally have per cell? And about how many cubic microns in volume is E. coli?

Answer 12

~3 molecules of lacR per cell
3 cubic microns
This corresponds to 1nM concentration of lacR, or 10^-9M. So, in E. coli, the operator will get occupied within 0.1 seconds. However, theoretically, the fastest a protein of that size and concentration can bind is 10 seconds, not 0.1 seconds. LacR binds to the operator 100x faster than possible by the laws of diffusion!

Question 13

Can RNAPs do facilitated diffusion?

Answer 13

Yes, but not as dramatic

Question 14

What is the most popular method to express proteins in E. coli?

Answer 14

The pET expression system

Question 15

In terms of the pET expression system, what can eliminate the ‘leakage’ issues (due to lacR dissociation)?

Answer 15

Using T7 and TWO operators

Question 16

What was the observation (known for 50+ years) about the formation of specific enzymes in bacteria? And what are the possible mechanisms?

Answer 16

Bacteria can form specific enzymes when the substrates for the enzymes (nutrients) are supplied
[The phenomenon is far more difficult to study in tissues or cells of higher organisms]
What is the mechanism? - enzyme adaptation? Gene regulation?*

Question 17

What did Jacob and Monod know in 1961?

Answer 17

Genes encode proteins, and genes are made of DNA. Positions of genes can be mapped genetically (frequency of recombination)

Question 18

What did Jacob and Monod not know in 1961?

Answer 18

• No concept of promoter
• Barely any concept of messenger RNA
• No concept of activation and CAP-based control; in hindsight, it was not a problem, as there was no glucose in their cell media anyway

Question 19

Why should you not confuse IPTG and ONPG?

Answer 19

IPTG is non-hydrolysable by beta-gal. Used to induce (switch on) expression of beta-gal
ONPG is hydrolysable by beta-gal. Used to measure the activity of beta-gal

Question 20

What does IPTG stand for?

Answer 20

Isopropyl-thio-galactoside

Question 21

What does ONPG stand for?

Answer 21

Ortho-nitrophenyl-beta-galactoside

Question 22

Are the genes for beta-gal (lacZ), permease (lacY), and trans-acetylase (lacA) close to each other on the chromosome or not?

Answer 22

They are very close to each other on the chromosome

They are also close to a gene called lacI

Question 23

What happens upon the addition of IPTG to the lacZ, lacY and lacA genes?

Answer 23

All 3 genes were induced together, at similar rates!

Therefore, the 3 genes must be ‘operated’ by a common switch hence the name ‘operon’! Polycistronic gene organisation

Question 24

In lacI mutants, what genes are ‘always on’, no matter if IPTG is there or not?

Answer 24

Permease and transacetylase

Question 25

What conclusion can be drawn about the lacI gene? And why?

Answer 25

It somehow controls all three genes at the same time - lacZ, Y and A
When IPTG is absent, lacI (or its product) represses all 3 genes lacZ, lacY and lacA at once
When IPTG is added, the repression by lacI (or its product) is lifted

Question 26

How does gene lacI control genes lacZ, Y and A?

Answer 26

The wild type gene on F’, lacI(+), makes a repressor, the repressor diffuses across the cell, and exerts normal off/on control

Question 27

What is the ability of the wild type repressor gene, lacI, to control genes located on a different DNA molecule called?

Answer 27

‘Regulation in trans’

Question 28

What does regulation in trans imply about lacI?

Answer 28

That lacI produces a diffusible product (the repressor) which then exerts the control

Question 29

How can you find out what is the operator?

Answer 29

Look for mutations in it, Oc
As expected, Oc had an ‘always on’ (constitutive) phenotype for lacZ, Y and A. However the O(-) mutation mapped not in lacI, but between lacI and lacZ! And, O(-) was dominant with respect to O(+), unlike lacI(-)

Question 30

What id the ability of the operator to control genes on the same DNA molecule called?

Answer 30

Regulation in ‘cis’

Question 31

What does regulation of the operon by the operator in ‘cis’ suggest?

Answer 31

That the operator is on the same DNA molecule as lacZ, Y, and A. I.e. it is most likely on the same fragment of DNA

Question 32

Oc mutations are mapped immediately downstream from what?

Answer 32

The promoter